Gap junctions between the follicle cells and the oocyte during oogenesis in an insect,Tribolium destructor/Coleoptera

Summary Oocyte-follicle cell gap junctions inTribolium occur in all oogenetic stages studied. During early previtellogenesis the junctions are found exclusively between lateral membranes of oocyte microvilli and the membrane of prefollicle cells. In late previtellogenesis and vitellogenesis the junctions are located between the tips of oocyte microvilli and the flat membranes of the follicle cells. During previtellogenesis gap junctions are infrequent, whereas in the phase of yolk accumulation their number increases considerably, exceeding 17 junctions/m² of the follicle cell membrane. It could be shown by microinjection of a fluorescent dye that gap junctions are in a functional state during vitellogenesis. Possible roles of heterologous gap junctions in oogenesis are discussed.     

Gap junctions and intercellular communication in the hepatopancreas of the crayfish (Orconectes propinquus) during molt

Summary The crustacean hepatopancreas is a major metabolic center intimately involved in molting and vitellogenesis. Cells of the hepatopancreas exhibit one of the richest endowments of gap junctions known and are thus presumed to be linked for intercellular communication. In order to monitor hepatopancreatic activity during the molt cycle of crayfish (Orconectes propinquus), the electrical coupling between cells of the hepatopancreatic tubules was measured during postmolt, intermolt and premolt. Samples of hepatopancreas from each of these stages were fixed and freeze-fractured to correlate morphologic features of gap junctions with electrophysiological data. Analysis of the data revealed that ionic coupling was greater in postmolt and premolt tubule cells than in cells of intermolt animals. Platinum replicas of hepatopancreatocyte plasmalemmata revealed that in postmolt, gap junction plaques were smaller and more numerous than those in intermolt and premolt; however, the total area of gap junction plaques per unit membrane area analyzed was approximately the same for hepatopancreatocytes from all molt stages. Although the hepatopancreatic gap junctions exhibited no quantitative differences, those from post- and premolt animals were rounded with tightly packed particles, while plaques from intermolt animals were generally pleomorphic with loosely packed particles. Results of this study suggest that cells of the crayfish hepatopancreas are more coupled in pre- and postmolt, with macular plaques of tightly packed particles, perhaps as a response to the increased metabolic demands of molt, and less well coupled, with irregular plaques of loosely packed junctional particles, during intermolt. The only recognizable morphological correlates of increased cell coupling were tight packing of junctional particles into rounded plaques, while decreased coupling corresponded to junctions with loosely packed irregular aggregates of particles.Supported by the Natural Sciences and Engineering Research Council of Canada (RRS)     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

Cell junctions in early embryos of squid (Loligo pealei)

Summary Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7–8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12–13). During germinal layer establishment (stages 12–13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavagestage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.     

Hybrids between normal cells,communication-deficient and communication-competent tumor cells

Summary Communication competent rat cells, communication incompetent (i.e., gap junction defective) rat tumor cells and communication competent rat tumor cells were fused in different combinations and the resultant hybrid cells were tested with regard to their growth properties and communication-competence. The ability to communicate via gap junctions was strictly inherited in a dominant fashion. Hybrids of normal cells and non-communicating tumor cells exhibited normal growth properties,i.e., the transformed phenotype was not expressed. This adds evidence to the hypothesis that the transformed growth of communication-incompetent tumor cells may be due to the loss or closure of their gap junctions.Communication-competent tumor cells behaved different when fused with normal cells in that their transformed growth was observed in each of the hybrids examined. No complementation was observed when different communication-incompetent cells or different tumor cells were fused.     

Role of the p38 MAP-Kinase Signaling Pathway for Cx32 and Claudin-1 in the Rat Liver

Liver regeneration and cholestasis are associated with adaptive changes in expression of gap and tight junctions through signal transduction. The roles of stress responsitive MAP-kinase, p38 MAP-kinase, in the signaling pathway for gap junction protein, Cx32, and tight junction protein, claudin-1, were examined in rat liver in vivoand in vitro, including regeneration following partial hepatectomy and cholestasis after common bile duct ligation. Changes in the expression and function of Cx32 and claudin-1 in hepatocytes in vivowere studied using the p38 MAP-kinase inhibitor SB203580. Following partial hepatectomy and common bile duct ligation, down-regulation of Cx32 protein was inhibited by SB203580 treatment. Up-regulation of claudin-1 protein was enhanced by SB203580 treatment after partial hepatectomy but not common bile duct ligation. However, no change of the Ki-67 labeling index (which is a marker for cell proliferation) in the livers treated with SB203580, was observed compared to that without SB203580 treatment. In primary cultures of rat hepatocytes, however, treatment with a p38 MAP-kinase activator, anisomycin, decreased Cx32 and claudin-1 protein levels. p38 MAP-kinase may be an important signaling pathway for regulation of gap and tight junctions in hepatocytes. Changes of gap and tight junctions during liver regeneration and cholestasis are shown to be in part controlled via the p38 MAP-kinase signaling pathway and are independent of cell growth.     

CONNEXIN EXPRESSION AND GAP JUNCTIONS IN THE MAMMARY GLAND

Gap junctional communication plays a vital role in embryogenesis, cell differentiation and the co-ordination of tissue responses. Gap junctions are formed by a family of closely-related proteins called connexins which show tissue-specific patterns of expression. The role of gap junctions in the mammary gland remains unclear. The lumena of mammary gland ducts are lined by luminal cells with an outer layer of basal cells. In rodents, the luminal cells express connexin26 only during pregnancy and lactation and the basal cells, in some reports, express connexin43. In the normal human breast the basal cells express connexin43, although human mammary epithelial cellsin vitrohave been reported to express both connexin26 and connexin43. Analysis of connexin expression at the molecular level is now bringing new insights into the structure and function of gap junctions in a range of normal and pathological cell systems.     

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein,in mammal heart and brain

Summary According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314–322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrathin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions.The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43.Proteins ofM _r 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.     

Reaching out: junctions between cardiac telocytes and cardiac stem cells in culture

Telocytes (TCs) were previously shown by our group to form a tandem with stem/progenitor cells in cardiac stem cell (CSC) niches, fulfilling various roles in cardiac renewal. Among these, the ability to ‘nurse’ CSCs in situ, both through direct physical contact (junctions) as well as at a distance, by paracrine signalling or through extracellular vesicles containing mRNA. We employed electron microscopy to identify junctions (such as gap or adherens junctions) in a co‐culture of cardiac TCs and CSCs. Gap junctions were observed between TCs, which formed networks, however, not between TCs and CSCs. Instead, we show that TCs and CSCs interact in culture forming heterocellular adherens junctions, as well as non‐classical junctions such as puncta adherentia and stromal synapses. The stromal synapse formed between TCs and CSCs (both stromal cells) was frequently associated with the presence of electron‐dense nanostructures (on average about 15 nm in length) connecting the two opposing membranes. The average width of the synaptic cleft was 30 nm, whereas the average length of the intercellular contact was 5 μm. Recent studies have shown that stem cells fail to adequately engraft and survive in the hostile environment of the injured myocardium, possibly as a result of the absence of the pro‐regenerative components of the secretome (paracrine factors) and/or of neighbouring support cells. Herein, we emphasize the similarities between the junctions described in co‐culture and the junctions identified between TCs and CSCs in situ. Reproducing a CSC niche in culture may represent a viable alternative to mono‐cellular therapies.     

Gap junctions between oocyte and follicle cells in Acerentomon sp. (Insecta,Protura)

Each oocyte in the ovary of Acerentomon is surrounded by a layer of follicle cells (FC) and possesses a group of specialized, so-called chorion-producing cells (CPC). The FCs lying immediately under the CPCs form processes which make contact with the oocyte. Gap junctions occur at the points of contact between the oolemma and the membrane of the processes. A possible role of the heterocellular gap junctions in Acerentomon ovary is the coordination of development of the oocyte and CPCs.     

Crustecdysone-induced modulation of electrical coupling and gap junction structure in crayfish hepatopancreatocytes

Crustecdysone, the hormone responsible for onset and regulation of the molt cycle in Crustacea, causes an increase in ionic coupling of cells of the hepatopancreas concomitant with the events of the molt. Hepatopancreatic tissue incubated for up to 4 hr in modified Eagle Basal Medium containing crustecdysone, exhibited an approximate 29% decrease in intercellular resistance as compared with tissue incubated in control medium. This represents a 29% increase in ionic coupling between hepatopancreatocytes following treatment with crustecdysone. Examination of platinum replicas of freeze-fractured, crustecdysone-treated hepatocyte plasma membrane revealed that most of the gap junction plaques were round with tightly packed intramembrane particles; a condition indicative of highly coupled cells. Similar preparations of control plasmalemmae demonstrated many gap junction plaques which were round or irregular in shape with very loosely packed particles and were indicative of uncoupled junctions. Results of this study are identical to those from a previous investigation of the electrophysiology and freeze-fracture morphology of hepatopancreatocytes during the molt cycle (McVicar and Shivers, 1984), and are thus presumed to reflect a crustecdysone-controlled increase in cell communications in vivo.     

Cell junctions during morphogenesis of feathers: general ultrastructure with emphasis on adherens junctions

Alibardi, L. 2011. Cell junctions during morphogenesis of feathers: general ultrastructure with emphasis on adherens junctions. —Acta Zoologica (Stockholm) 92 : 89–100. The present ultrastructural and immunocytochemical study analyzes the cell junctions joining barb/barbule cells versus cell junctions connecting supportive cells in forming feathers. Differently from the epidermis or the sheath, desmosomes are not the prevalent junctions among feather cells. Numerous adherens junctions, some gap junctions and fewer tight junctions are present among differentiating barb/barbule cells during early stages of their differentiation. Adherens junctions are frequent also among differentiating supportive cells and show weak immunolabeling for both N‐cadherin and neural‐cell adhesion molecule (N‐CAM). Differentiating barb and barbule cells do not show labeled junctions for N‐cadherin and N‐CAM. The labeling occurs at patches in the cytoplasm of supportive cells but is more frequently seen in the external cytoplasm and along the extra‐cellular space (glycocalix) covering the plasma membrane of supportive cells. Labeling for N‐cadherin is also found in medium‐dense 0.1‐ to 0.3‐μm granules present in supportive cells and sometimes is associated with coarse filaments or periderm granules. The study indicates that adherens junctions form most of the transitional connections among supportive cells before their degeneration. Keratinizing barb and barbule cells loose the labeling for adherens junctions (N‐CAM and N‐chaderin) while their adhesion is strengthened by the incorporation of cell junctions in the corneous mass forming the barbules.     

Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

Passage through vertebrate gap junctions of 17/18 kDa molecules is primarily dependent upon molecular configuration

In fish, amphibians and mammals, gap junctions of some cells allow passage of elongate molecules as large as 18 kDa, while excluding smaller, less elongate molecules. Fluorescently labeled Calmodulin (17 kDa) and fluorescently labeled Troponin-C (18 kDa), when microinjected into oocytes of Danio rerio, Xenopus laevis or Mus domestica, were able to transit the gap junctions between these oocytes and the granulosa cells which surrounded them. Co-microinjected with these Ca²⁺-binding proteins, Texas-red-labeled dextran (10 kDa) remained in the microinjected cell. Osteocalcin (6 kDa), also a Ca²⁺-binding protein, but with a wide “V” shape proved unable to transit these gap junctions. Calmodulin, but not Troponin-C, was able to transit gap junctions of gonadotropin treated WB cells in culture. We show evidence that molecules as large as 18 kDa can pass through some vertebrate gap junctions, both homologous and heterologous, and that it is primarily molecular configuration which governs gap junctional permeability.     

Association between mitochondria and gap junctions in mammalian myocardial cells

In ventricular myocardial cells of mouse, guinea-pig, dog, and monkey, mitochondria frequently form close associations with gap junctions, the two structures being separated by a space of 20 nm or less. Similar appositions are found in both the mature atria and the developing myocardium of the mouse. The gap junctions assume a variety of configurations with respect to the apposed mitochondria. These include profiles in which the gap junctions conform closely to the contours of mitochondria, as well as profiles in which finger-like sarcolemmal evaginations, composed entirely of gap junctions, extend longitudinally or transversely into an adjoining cell to envelop mitochondria. In mouse ventricular wall, over 40% of the length of gap junctions is juxtaposed to mitochondria, and strands of connecting material are often present in the interspace between the two structures. In addition, in freeze-fracture replicas, portions of mitochondria are found attached to areas of myocardial sarcolemma that contain gap-junctional particles. Since mitochondria are known to sequester Ca²⁺ ion, it is possible that the close association between mitochondria and gap junction may function to buffer the intracellular Ca²⁺ concentration near the gap junctions, and thereby regulate the ionic permeability of the gap junctions.     

Gap junctions of the muscles of the small and large intestine

Summary The distribution of gap junctions (nexuses) in various parts of the small and large intestines of the guinea-pig was studied using the freeze-fracture technique and in thin sections. The percentage area of smooth muscle cell surface occupied by gap junctions varies from 0.50% in the circular muscle of the duodenum to zero in the longitudinal muscle of the ileum. In the circular muscle of the jejunum and ileum the area occupied by nexuses is 0.22% (or about 11 m² per cell). The sizes of junctions range from less than 0.01 m² to 0.20 m², with two-thirds of them being smaller than 0.05 m². In the colon, gap junctions are rare, very small and confined to the circular muscle layer. Even the smallest aggregates of intramembrane particles correspond to areas of close apposition between the membranes of adjacent cells; it is therefore justified to interpret them as being gap junctions. Some gap junctions are formed between a smooth muscle cell and an interstitial cell. Gap junctions are not found in the longitudinal muscle of the small intestine; this is in sharp contrast to the abundance of gap junctions in the adjacent circular layer.In the small intestine of cats and rabbits, gap junctions are abundant in the circular muscle layer, whereas they are very small in size and very few in number in the longitudinal muscle layer.The authors wish to thank Mr Peter Trigg and Miss Eva Franke for help and support. This work was supported by grants from the Medical Research Council and the Central Research Fund of the University of London     

Cells and intercellular contacts in glomeruli and tubules of the frog kidney

Summary By the use of thin sections and freeze-fracture replicas the glomerular and tubular structures of the kidney of the frog (Rana esculenta) were studied with special reference to intercellular junctions.In the glomerulus the filtration barrier is of very variable thickness, and frequent tight and gap junctional contacts occur between podocyte processes.Although structurally less elaborate, the proximal tubule resembles its mammalian counterpart. In the initial part the tight junctions are relatively shallow but become very broad in the mid and distal portions of the proximal tubule. The proximal tubular cells are extensively linked by gap junctions. In some animals the shapes of the cells in the proximal and distal portions of the proximal tubule were markedly different.The distal tubule consists of two segments which differ mainly in the pattern of interdigitations and the structure of the zonulae occludentes. Similarities with the tight junctional morphology of the mammalian distal tubule are striking. In the first part of the distal tubule (diluting segment) a narrow band of parallel tight junctions is found closely resembling that found in the mammalian straight distal tubule; in the more distal part of the distal tubule, however, a broad band of anastomosing tight junctional strands exists, like the zonula occludens of the mammalian convoluted distal tubule.The connecting tubule displays cellular dimorphism: its wall contains a mixture of light and dark (flask) cells. The luminal and basolateral membranes of the flask cells are covered with numerous rod-shaped particles. The tight junctions of the connecting tubule are broad and increase in depth and number of strands along its length; they are typical of a very tight epithelium.In spite of several dissimilarities with phylogenetically younger kidneys our findings suggest that many structural principles of the mammalian kidney are also represented in the kidneys of amphibians. The structural-functional relationships are discussed.     

Cell junctions in the rapidly moving edge of chick and quail blastoderms during gastrulation

Summary The intercellular contacts of the migrating edge of chick and quail blastoderms during gastrulation were studied by transmission electron microscopy of thin sections and of freeze-fracture replicas. Tight junctions and gap junctions as well as desmosomes were found. Tight junctions were organized as single junctional strands or as a complex of numerous junctional strands interposed between the lamellae and the bodies of the cells building up the margin of overgrowth. The function of these intercellular junctions is considered in relation to the locomotion of the margin of overgrowth cells.     

Intercellular junctions of antennal gland epithelial cells in the crayfish,Orconectes virilis

Summary Labyrinth and nephridial canal cells of the crayfish (Orconectes virilis) antennal gland possess two types of intercellular junctions revealed by freeze-fracture studies. Apical margins of the cells are connected by long septate junctions. In replicas, these junctions consist of many parallel rows of 80–140 Å intramembrane particles situated on the PF membrane face (EF and PF fracture faces of Branton et al., 1975). Rows of pits are found on the EF fracture face and are deemed complementary to the rows of particles. Moreover, lateral margins of basal regions of the epithelial cells are attached by many intercellular junctions. These contacts are characterized in thin plastic sections by a narrow dense cytoplasmic plaque located subjacent to the plasma membrane at sites of adjoined cells, and 5 to 12 fine strands of dense material that extend across the intercellular gap between adjoined cells. In freeze-fracture replicas, EF intramembrane faces basal to the region of the plasma membrane containing septate junctions exhibit numerous discoid clusters of particles. The particle aggregates, assumed to represent freeze-cleave images of adhering junctions, range from 900 to 3,700 Å in diameter, with individual particles about 185 Å in diameter. These junctions appear to connect epithelial cell processes formed by basal infoldings of the plasma-lemma, and occur between adjacent cells as well as adjacent processes of a single cell. The discrete aggregates of particles resemble replicated desmosomes (Shienvold and Kelly, 1974) and hemi-desmosomes (Shivers, 1976); therefore, they probably do not constitute a basis for electrical coupling between antennal gland epithelial cells.Supported by the National Research Council of Canada